Ludmila Fadeev

Formation of a Soluble Stable Complex between Pristine C60‐Fullerene and a Native Blood Protein

Concern is growing about the potential impact of human exposure to carbonaceous nanomaterials (such as fullerenes) in the environment. A valid biological study of how native biomolecules interact with nanomaterials at the molecular level in physiological conditions requires the preservation of their physicochemical properties, yet most investigations rely on the use of modified fullerene conjugates or aggregates. We report the formation of a stable, water‐soluble, well‐defined complex between a single molecule of pristine C60‐fullerene and a native protein, bovine serum albumin protein (BSA), with the normal three‐dimensional structure of BSA preserved. The ability to produce a pristine C60‐fullerene–BSA hybrid at a physiological pH range lays a solid foundation for studying carbonaceous materials, biodelivery systems, and transport mechanisms and for characterizing the potential effects of nanomaterials on wildlife and human health, both in vitro and in vivo.

In vitro synthesis of uniform poly(dG)–poly(dC) by Klenow exo− fragment of polymerase I

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo− fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.

Mucin complexes of nanomaterials: first biochemical encounter.

In recent years, the exposure of biological systems to various nanomaterials has become an issue of great public concern. Although living organisms have arrays of biological defense mechanisms against exposure to exogenous compounds, the biochemical mechanisms allowing various nanomaterials to enter the body are not well understood. A unique example of a typical mucosal glycoprotein capable of binding and solubilizing nanomaterials in physiological solution is provided, suggesting a possible route for entry into biological systems.

Enhanced bioavailability of polyaromatic hydrocarbons in the form of mucin complexes.

Increasing exposure of biological systems to large amounts of polycyclic aromatic hydrocarbons is of great public concern. Organisms have an array of biological defense mechanisms, and it is believed that mucosal gel (which covers the respiratory system, the gastrointestinal tract, etc.) provides an effective chemical shield against a range of toxic materials. However, in this work, we demonstrate, for the first time, that, upon complexation of polyaromatic hydrocarbons with mucins, enhanced bioavailability and, therefore, toxicity are obtained. This work was aimed to demonstrate how complexation of various highly hydrophobic polycyclic aromatic hydrocarbons with representative mucin glycoprotein could lead to the formation of previously undescribed materials, which exhibit increased toxicity versus pristine polycyclic aromatic hydrocarbons. In the present work, we show that a representative mucin glycoprotein, bovine submaxillary mucin, has impressive and unprecedented capabilities of binding and solubilizing water-insoluble materials in physiological solution. The complexes formed between the mucin and a series of polycyclic aromatic hydrocarbons were comprehensively characterized, and their toxicity was evaluated by both in vivo and in vitro assays. In addition, the bioavailability and membrane-penetration capabilities were tested using an internalization assay. Our results provide, for the first time, evidence of an unknown route by which hydrophobic materials may achieve higher bioavailability, penetrating some of the biological defense systems, in the form of water-soluble complexes with mucosal proteins.

Biodelivery of a fullerene derivative.

Most current nanotoxicology research is focused on examining the influence of nanomaterials at the tissue and cellular levels. To explore these interactions on the molecular level, new carboxyfullerenes interact with transport proteins at the molecular level. The carboxyfullerenes exhibited an unusual mode of binding outside the calyx of beta-lactoglobulin (a typical representative of lipocalin family of barrier liquid proteins). The complexes were studied by various techniques, including mass spectrometry, UV/vis and circular dichroism spectroscopy, chromatographic methods, gel electrophoresis, and dynamic light scattering. The fullerene ligands were transferred from beta-lactoglobulin to human serum albumin (a representative of a blood transport protein), thus providing a model of how fullerene-based nanomaterials interact with biomolecules and are transported in biological systems.

Surface-Induced Conformational Changes in Doped Bovine Serum Albumin Self-Assembled Monolayers

Evidence for considerable stabilization of doped bovine serum albumin (BSA) molecules upon adsorption on gold surfaces is provided. This is compared to the surface-induced conformational changes of the bare BSA and its corresponding monolayer. The BSA unfolding phenomenon is correlated with dehydration, which in turn enables improved monolayer coverage. The stabilization mechanism is found to be partially controllable via nanodoping of the BSA molecules, upon which the dehydration process is suppressed and molecular rigidity can be varied. Our experimental data and calculations further point to the intermixing of structural characteristics and inherent molecular properties in studies of biological monolayers.

Diameter-selective dispersion of carbon nanotubes by β-lactoglobulin whey protein

The β-lactoglobulin (β-LG) protein was discovered to be an efficient and selective dispersant for carbon nanotubes (CTNs) with certain diameters. A dispersion process of CTNs by the β-LG was studied, focusing on the relationships between the surface curvature of the CNT and the β-LGs efficiency in dispersing them, using cryogenic-transmission electron microscopy (cryo-TEM) and optical spectroscopy. Plausible binding sites of the β-LG, responsible for the interaction of the protein with CNTs of various diameters (surface curvatures) were also investigated and were found to be in good agreement with corresponding docking calculations.

The impact of highly hydrophobic material on the structure of transferrin and its ability to bind iron

Transferrin is a blood-plasma glycoprotein, which is responsible for ferric-ion delivery and which functions as the most important ferric pool in the body. The reversible complexation process of Fe(3+) ions is associated with conformational changes of the three-dimensional structure of the transferrin. This conformational dynamics is attributed to a partial unfolding of the N-lobe of the protein and could be described as a transition between the holo to the apo forms of the transferrin. The aim of the present work is to demonstrate the unprecedented ability of the transferrin to solubilize various polycyclic aromatic hydrocarbons in physiological solution and to explore the impact of these materials on the structure and functionality of the transferrin. The synthesis and characterization of novel materials, consisting of complexes between human transferrin and hydrophobic high-carbon-content compounds, is reported here for the first time. Furthermore, it is shown that the preparation of these complexes from holo-transferrin leads to an irreversible loss of the ferric ions from the protein. Analytical studies of these novel complexes may shed a light on the mechanism by which transferrin could lose its ability to bind and thus to transport and store iron. These findings clearly demonstrate a possible damaging impact of various hydrophobic pollutants, which can enter an organism by inhalation or ingestion, on the functionality of the transferrin.

Macroalgal biomass subcritical hydrolysates for the production of polyhydroxyalkanoate (PHA) by Haloferax mediterranei

Non-conventional carbon sources, such as macroalgae, are sustainable alternatives for large-scale production of biopolymers. The present study examined macroalgae-derived carbohydrates, as carbon sources for the production of polyhydroxyalkanoates (PHAs) by Haloferax mediterranei. Simulants of the hydrolysates of seven different macroalgal biomasses were prepared and the PHA production was studied. A maximum biomass concentration with maximum PHA content was detected in medium prepared from green macroalgae. The highest cell dry weight and PHA concentrations were 3.8 ± 0.2 g·L-1 and 2.2 ± 0.12 g·L-1 respectively when Haloferax mediterranei was grown in 25% (w/w) of Ulva sp. hydrolysate, at 42 °C temperature and initial pH of 7.2. Poly(3-hydroxy-butyrate-co-3-hydroxyvalerate was the major PHA constituent. The present study demonstrated that Ulva sp. is a promising feedstock for PHA production.

Silver Nanoparticles Complexed with Bovine Submaxillary Mucin Possess Strong Antibacterial Activity and Protect against Seedling Infection

ABSTRACT A simple method for the synthesis of nanoparticles (NPs) of silver (Ag) in a matrix of bovine submaxillary mucin (BSM) was reported previously by some of the authors of this study. Based on mucin characteristics such as long-lasting stability, water solubility, and surfactant and adhesive characteristics, we hypothesized that these compounds, named BSM-Ag NPs, may possess favorable properties as potent antimicrobial agents. The goal of this study was to assess whether BSM-Ag NPs possess antibacterial activity, focusing on important plant-pathogenic bacterial strains representing both Gram-negative (Acidovorax and Xanthomonas) and Gram-positive (Clavibacter) genera. Growth inhibition and bactericidal assays, as well as electron microscopic observations, demonstrate that BSM-Ag NPs, at relatively low concentrations of silver, exert strong antimicrobial effects. Moreover, we show that treatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedling transmission of Acidovorax citrulli, one of the most threatening pathogens of cucurbit production worldwide. Overall, our findings demonstrate strong antimicrobial activity of BSM-Ag NPs and their potential application for reducing the spread and establishment of devastating bacterial plant diseases in agriculture. IMPORTANCE Bacterial plant diseases challenge agricultural production, and the means available to manage them are limited. Importantly, many plant-pathogenic bacteria have the ability to colonize seeds, and seed-to-seedling transmission is a critical route by which bacterial plant diseases spread to new regions and countries. The significance of our study resides in the following aspects: (i) the simplicity of the method of BSM-Ag NP synthesis, (ii) the advantageous chemical properties of BSM-Ag NPs, (iii) the strong antibacterial activity of BSM-Ag NPs at relatively low concentrations of silver, and (iv) the fact that, in contrast to most studies on the effects of metal NPs on plant pathogens, the proof of concept for the novel compound is supported by in planta assays. Application of this technology is not limited to agriculture; BSM-Ag NPs potentially could be exploited as a potent antimicrobial agent in a wide range of industrial areas, including medicine, veterinary medicine, cosmetics, textiles, and household products.